Common and diverse mechanisms of pattern formation of animals

1)Tbx 2/3/4/5基因复合体与脊椎动物肢体和心脏形态的共同进化我们发现Tbx 5和Tbx 4基因在鸡胚的翼床和腿床之间的侧翼区域有错误表达。出乎意料的是，这种错误表达分别通过tbx 5和tbx 4诱导了完全的翅芽和腿芽。与这些观察结果相一致，在预期的翅膀和腿领域中Tbx 5和Tbx 4的显性负性形式的过度表达分别导致翅膀和腿结构的完全丧失。这些结果表明，Tbx 5和Tbx 4是翼和腿芽形成的启动子，最多位于lib形成信号通路的上游，并且Tbx 5在发育中的小鼠和鸡心脏的左心室中特异表达，提示Tbx 5可能参与左右心室的特化。为了证实这种可能性，我们在鸡心脏中错误表达了Tbx 5。当Tbx 5在整个心室表达时，室间隔不表达， ...更多信息 未形成。当Tbx 5在右心室表达时，室间隔位置发生偏移。这些结果表明，Tbx 5基因表达的区域决定了左心室的性质，在Tbx 5基因表达的边界形成了室间隔。在发育的肢芽中，Tbx 2和Tbx 3在肢芽的后部表达，其中Tbx 2在最后部表达，Tbx 3在更靠前的部分表达，提示这些基因可能参与了后趾的特化。为了证实这一点，我们在鸡肢芽中错误表达了这些基因.正如预期的那样，这些基因的错误表达诱导后验的数字身份。这些基因的抑制诱导前向转化，具有类似于人类尺乳综合征的典型表型。2）小脑发育和Irx 2。我们克隆了6个鸡Irx基因，发现其中一个Irx 2基因在小脑发育的菱形唇中特异表达。当Irx 2在中脑过表达时，没有获得形态学变化，表明另一个未知因子与Irx 2协同调节小脑发育。当Irx 2和FGF 8在中脑中共表达时，观察到中脑向小脑的完全转化。生物化学分析显示Irx 2被MAP激酶磷酸化，MAP激酶被FGF 8激活。这些数据表明Irx 2是小脑形成的关键决定因素之一，与FGF 8/MAP激酶的组织者活性协同作用。3）机械应力、转录调控和发育。在寻找Tbx 5的共激活因子时，我们已经确定了几种协同激活ANF（心钠素）启动子（Tbx 5的直接靶点）的因子。其中之一，MKL 2，反式激活ANF几百倍。有趣的是，这种激活被RhoA和非经典Wnt信号强烈增强。在斑马鱼心脏亚细胞定位分析中，发现MKL 2在跳动心脏中定位于细胞核，而在无跳动心脏中则定位于细胞核。这表明MKL 2的定位是由机械应力控制的，例如张力和剪切应力诱导的心跳。与此相适应，MKL 2响应于培养细胞的机械拉伸而穿梭进入细胞核。因此，我们得出结论，Tbx 5的转录活性是受机械应力依赖的核质穿梭MKL 2。这也表明心脏发育受遗传和表观遗传程序的调节，其中血液动力学的物理参数起着重要作用。除了MKL 2，我们还鉴定了几种穿梭因子。我们的初步数据表明，这些因素调节间充质干细胞的分化，脂肪生成与肌生成，脂质代谢和动态形态发生细胞的行为，突出机械应力作为一个关键因素，参与了各个方面的发展和稳态。少

1) Co-evolution of the Tbx2/3/4/5 gene complex and morphology of vertebrate limb and heart. We mis -expressed Tbx5 or Tbx4 gene in flank regions between wing and leg beds of chick embryos. Unexpectedly, such mis -expression induced complete wing and leg buds by tbx5 and tbx4, respectively. Compatible with these observations, over-expression of the dominant negative forms of Tbx5 and Tbx4 in the prospective wing and leg fields resulted in a complete loss of wing and leg structures, respectively. These data indicate that Tbx5 and Tbx4 is the initiator of wing and leg bud formation, lying at the most, upstream of the putative signaling pathway of lib formation.We also found that Tbx5 is specifically expressed in the left ventricle of developing mouse and chick hearts, implying that Tbx5 might be involved in specification of left and right ventricles. To confirm this possibility, we mis-expressed Tbx5 in chick hearts. When Tbx5 was expressed in the whole ventricle, ventricular septum was n … More ot formed. When Tbx5 was expressed in the right ventricle, position of septum was shifted. These indicate that Tbx5 specifies the identity of left ventricle, and that the ventricular septum was formed at the boundary of Tbx5 expression. In addition, we identified Tbx20 was a right ventricle-specific Tbx gene.In the developing limb buds, Tbx2 and Tbx3 are expressed in posterior part of limb bud, with Tbx2 in the most posterior part and Tbx3 in a more anterior, suggesting that these genes might be involved in specification of posterior digits. To confirm this, we mis -expressed these genes in chick limb buds. As expected, misexpression of these genes induces posteriorization of digit identity. Suppression of these genes induces anterior transformation, with a typical phenotype that resembles that of human ulnar-mammary syndrome.2) Cerebellum development and Irx2. We cloned 6 chick Irx genes and found that one of them, Irx2, is specifically expressed in the rhombic lip, from which cerebellum develops. When Irx2 was over-expressed in midbrain, no morphological change was obtained, indicating that another unknown factor(s) cooperate with Irx2 to regulate cerebellar development. When Irx2 and FGF8 were co-expressed in the midbrain, complete transformation of midbrain to cerebellum was observed. Biochemical analyses revealed that Irx2 is phosphorylated by MAP kinase, which is activated by FGF8. These data indicate that Irx2 is one of critical determinant of cerebellum formation, cooperating an organizer activity of FGF8/MAP kinase.3) Mechanical stresses, transcriptional control and development. In search of co-activator(s) of Tbx5, we have identified several factors that synergistically activate ANF (atrial natriuretic factor) promoter, a direct target of Tbx5. One of them, MKL2, transactivates ANF several hundred fold. Interestingly, this activation was strongly enhanced by RhoA and non-canonical Wnt signaling. When sub-cellular localization of MKL2 was analyzed in zebrafish heart, MKL2 was found to be in nucleus in beating heart, whereas in non-beating silent heart. This indicates that localization of MKL2 was controlled mechanical stresses, such as tension and sheer stresses induced heartbeat. Compatible with this, MKL2 shuttles into nucleus in response to mechanical stretch of cultured cell. Hence, we conclude that transcriptional activity of Tbx5 is regulated by mechanical stress-dependent nucleo-cytoplasmic shuttling of MKL2. This also suggests that cardiac development is regulated by the genetic and epigenetic programs, in which physical parameters of hemodynamics play important roles. In addition to MKL2, we have identified several shuttling factors. Our preliminary data indicate that such factors regulate differentiation of mesenchymal stem cells, adipogenesis vs. myogenesis, lipid metabolism and dynamic morphogenetic cell behaviors, highlighting mechanical stresses as a critical factor that is involved in various aspects of development and homeostasis. Less